Role of factors downstream of caspases in nuclear disassembly during apoptotic execution

We used cytoplasmic extracts from chicken DU249 cells at various stages alo ng the apoptotic pathway to analyse the events of apoptotic execution. So-c alled S/M extracts from morphologically normal 'committed-stage' cells indu ce apoptotic morphology and DNA cleavage in substrate nuclei. These apoptot ic changes appear to require the function of multiple caspases (cysteine as partases, a specialized class of proteases) acting in parallel. Extracts fr om 'execution-stage' apoptotic cells induce apoptotic events in added nucle i in a caspase-independent manner. Biochemical fractionation of these extra cts reveals that a column fraction enriched in endogenous active caspases i s unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. 'Executi on-stage' extracts contain an ICAD/DFF45-inhibitable nuclease resembling CA D, plus another activity that is required for the apoptotic chromatin conde nsation. 'Committed-stage' S/M extracts lack these downstream activities. T hese observations reveal that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than di sassembling it themselves. They also suggest that activation of the downstr eam factors (rather than the caspases) is the critical event that occurs at the transition from the latent to the execution phase of apoptosis.